Fungal infections are a particular health care concern in immunocompromised patients, including patients with acquired immune deficiency syndrome (AIDS) where opportunistic infections such as Pneumocystis carinii are responsible for significant morbidity and mortality. In the 1988 revision of the definition of AIDS by CDC and the World Health Organization (WHO), candidosis of the esophagus, trachea, bronchi, or lungs and meningeal cryptococcosis were recognized as important "indicator" discases. In the presence of laboratory evidence of HIV infection, disseminated coccidioldomycosis and histoplasmosis are also considered indicative of AIDS. It has been estimated that 58-81% of all AIDS patients contract a fungal infection at some time during the prodromal stage or after developing AIDS, and that 10-20% have died as a direct consequence of fungal infections. Major mycoses related to AIDS include candidosis, cryptococcosis (yeasts), histoplasmosis, and coccidioidomycosis (dimorphic fungi). Deep, severe, but relatively rare mycoses related to AIDS include penicilliosis, blastomycosis, paracoccidioidomycosis, sporotrichosis, aspergillosis, mucormycosis, yeast infections, and nocardiosis.
Aspergillosis, while less common in AIDS patients, is a common fungal infection in other immunodepressed patients, occurring at a rate as high as 70% in patients with leukemia after 30 days of neutropenia. Cutaneous fungal infections elated to AIDS include sebortheic dermatitis, dermatophytosis, trichosporonosis, and alteraariosis.
In addition to the, pathological damage resulting directly from fungal infections, fungal antigens can act as immunosuppressors which may play a role as a cofactor in the development of AIDS. While small amounts of fungal antigen can stimulate the immune response, an excess of antigen may have an adverse effect on cellular immunity. Fungal circulating antigens, such as mannan in candidosis, and glucuronoxylomannan in cryptococcosis may be present in excess in acute fungal infections. Candidal antigens may be important cofactors in AIDS. It is imperative that treatment must be undertaken rapidly and efficiently before these conditions lead to invasive forms. The immunosuppressive effect of other fungal antigens by induction of T-suppressor activity has also been proposed.
Systemic fungal infections are difficult to diagnose antemortem in AIDS patients. Unfortunately, autopsy is often the only available route to diagnose fungal infections. AIDS patients with fungal infections may have nonspecific symptoms over a long period of time. It has been difficult to establish definitive diagnoses from patients' body fluids. Histologic identification of organisms requires invasive procedures, with possible attending complications. Isolation of the organisms in blood culture, when possible, can sometimes be used for diagnosis. Even so. proper diagnosis is delayed because of the time required to process the specimens and culture the fungus. This delay alone can result in progressive deterioration from the disease. Some AIDS patients with fungal infections respond to appropriate therapy quickly with early diagnosis, although continued lifetime therapy may be necessary due to their underlying abnormal immune systems.
To improve the care of AIDS and other immunocompromised patients, a mechanism for the early diagnosis of fungal infections is imperative. Prompt implementation of an appropriate antifungal therapy provides a better environment for antiviral chemotherapy. Despite recent advances in anti-fungal therapeutics, which show promising efficacy for many of the mycoses, a rapid, sensitive, and accurate fungal diagnostic method which is also quantitative and broad-spectrum is still unavailable.
Chitin is a substance common to most fungi. Chitin is a class of polymers of N-acetyl-glucosamine. Chitin and glucan are the major constitents of cell walls of most fungi, and of some yeasts. It has been observed that the cell wall composition of a fungus is not always fixed, and may vary during the life cycle in a single species. Recent identification of chitin in certain stages in Oomycetes is an example.
Many diagnostic methods presently available for fungi are designed to detect specific anti-fungal antibodies in body fluids such as blood or serum, for example, anti-candida albicans, anti-cryptococcus, anti-histoplasma, anti-blastomyces, anti-aspergillus and anti coccidioidomyces. These tests include immunodiffiusion, latex antibody agglutination, complement fixation and candida enzyme immunoassay.
The only currently available quantitative diagnostic test for circulating fungal antigen, Cyptcoccus latex agglutination, is limited to detection of Cryptococcus. For a reliable result, treatment of the sample with a protease, "Pronase," is necessary. Due to the low sensitivity of each individual test (e.g., about 80% for an immunodiffusion test), a single assay is often not definitive.
There are other available methods for the selective histologic identification of fungal organisms in tissue specimens, each of which has disadvantages. These methods generally have a broader sensitivity than antibody detection methods, in that they can recognize more than one species of fungi. Their common disadvantages are difficulties when applied to samples of body fluids, because proper sample fixation can be difficult, and their inability to give good quantitative results. Grocott methenamine silver nitrate (GMS) stain is by far the most common currently used method in the pathology laboratory. GMS staining takes advantage of the presence of polysaccharides in most fungal organisms to create a contrasting image between the fungus and the host tissue. The stain is not as effective when it is used in the cytospin or other samples of body fluids. GMS staining can be non-specific, due to its indiscriminate recognition of connective tissue polysaccharides (e.g. glycosaminoglycans, and mucin), and is not quantitative. Other histochemical stains for fungal organisms include calcofluor/cellufluor, India Ink, lectin label, and Rylus BSU. Some of the specific disadvantages of the individual methods are the following:
Calcofluor/Cellufluor: This method uses calcofluor/cellufluor to bind to the chitin of fungal organisms, which will then self-fluoresce under ultraviolet light. This method requires fluorescence microscopy, which limits its use in small clinics. PA1 India Ink: This method uses India Ink to detect capsulated organisms such as Cryptococci, but is limited because it detects only cells with such a capsule. Many fungi do not have such a capsule. PA1 Lectin label: Lectin label has been used in the past to stain fungi with a specific lectin-binding property (e.g., wheat germ agglutinin). However, non-specific staining is problematic. PA1 Rylus BSU: this method stains the chitinous cell walls of fungal organisms, which will then self-fluoresce under ultraviolet light. This method requires fluorescence microscopy, which limits its use in small clinics. PA1 Chamberland et al., "Chitinase-Gold Complex Used to Locaize Chitin Ultrastructurally in Tomato Root Cells," Histochem. J., Vol. 17, pp 313-321 (1985), discusses the use of a fungal-extracted chitinase conjugated with gold to detect chitin in a Fusarium oxysporum infection of tomato root cells. Detection was performed with an electron microscope. Electron microscopy techniques are expensive, and would be impractical in many clinics and hospitals. This reference discusses only plants. Nothing in it suggests the use of a chitinase or other chitin-specific binding protein for the diagnosis of fungal or yeast infections in animal or human tissues or body fluids. See also Benhamou, et al, "Attempted Loalization of a Substrate for Chitinases in Plant Cells Reveals Abundant N-acetyl-D-glucosamine Residues in Secondary Walls," Biology of the Cell, vol. 67, No. 3, pp. 341-50 (1989).
For the above reasons and others, thee exists an unfulfilled need for an improved method of diagnosing fungal infections, especially in animals and humans, either in tissue samples or in body fluids.